Fusion proteins comprising antigenic apob-100 protein fragments and a protein carrier, related compositions, methods and systems for treatment and/or prevention of atherosclerosis

ABSTRACT

A fusion protein comprising an antigenic fragment of apoB-100 and a suitable carrier and related compositions methods and systems.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent application Ser. No. 13/021,635, filed Feb. 4, 2011 which claims priority to U.S. provisional application 61/302,051 filed on Feb. 5, 2010, each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to fusion proteins and related compositions, methods and systems for treatment and/or prevention of atherosclerosis.

BACKGROUND

Atherosclerosis is currently viewed as a chronic lipid-related and immune-mediated inflammatory disease of the arterial walls. Many immune components have been identified that participate in atherogenesis and pre-clinical studies have yielded promising results suggesting that immunomodulatory therapies targeting these components can reduce atherosclerosis.

SUMMARY

Provided herein, are methods and systems for inducing immunomodulatory responses in an individual. In several embodiments, the immunomodulatory responses induced by the methods and systems of the present disclosure are associated to a therapeutic or preventive effect related to atherosclerosis in the individual or a condition associated thereto.

According a first aspect a fusion protein is described. The fusion protein comprises an antigenic fragment of apoB-100 protein or a derivative thereof and a protein carrier, the antigenic fragment and the carrier comprised in the fusion protein in a fragment:carrier 1:1 molar ratio the fusion protein capable of inducing antigen-specific regulatory T cells, the antigen-specific regulatory T cells specific for the antigenic fragment of apoB-100.

According to a second aspect, a method to treat and/or prevent atherosclerosis in an individual is described. The method comprises administering to the individual an effective amount of a fusion protein herein described, the effective amount eliciting an antigen-specific T regulatory immunomodulatory response in the individual, the antigen-specific Treg immunomodulatory response specific for the antigenic fragments of apoB-100 or a derivative thereof.

According to a third aspect, a composition is described. The composition comprises a fusion protein herein described and an adjuvant and/or excipient. In several embodiments the adjuvant and/or excipients are pharmaceutically acceptable and the composition is pharmaceutical composition

According to a fourth aspect, a method to produce a fusion protein is described. The method comprises conjugating a fragment of apoB-100 or a derivative thereof with a suitable protein carrier to provide a fusion protein capable of inducing antigen-specific regulatory T cells, the antigen-specific regulatory T cells specific for the fragment of apoB-100 or the derivative thereof.

According to a fifth aspect, a method to induce an antigen-specific Tregulatory cell is described. The method comprises contacting a Tregulatory cell with a fusion protein herein described for a time and under conditions to allow induction of a Tregulatory response, wherein the contacting results in an antigen-specific induction of a Tregulatory cell that is specific for the fragment of apoB-100 or derivative thereof comprised in the fusion protein.

The methods and systems herein described can be used in connection with applications wherein reduction of plaque, attenuation of plaque growth and/or a therapeutic or preventive effect for atherosclerosis in an individual is desired.

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and examples sections, serve to explain the principles and implementations of the disclosure.

FIG. 1 shows data concerning intranasal p210-CTB administered twice weekly for 12 weeks reduced atherosclerotic lesion size in the aortic root of female apoe^(−/−) mice. (A) Data from the three groups are shown: black circles represent animals from the p210-CTB group, grey circles animals from the OVA-CTB group and white circles animals from the control group, respectively. * indicates p<0.05. (B-D) Representative photomicrographs show oil red 0 stained aortic root sections from each group (50× magnification). (E) Nasal immunization with CTB fusion proteins increased FoxP3 and IL-10 mRNA levels in thoracic aorta from apoe^(−/−) mice after 12 weeks of treatment. mRNA transcript ratios based on HPRT expression are shown for each gene of interest for all three groups. * indicates p<0.05

FIG. 2 shows data nasal immunization induced a systemic humoral immune response in apoe^(−/−) mice. (A) IgG-anti-p210 titers in mouse plasma; titration curves are shown in the inset. (B) p210-specific IgM titers in plasma from the same mice. * indicates p<0.05.

FIG. 3 shows data concerning nasal immunization altered T cell subset composition in lung mucosa. Flow cytometric analysis of intracellular subset markers, with cytokine-producing CD4⁺ T cells as percentage of total CD4⁺ T cells for each of the three groups. (A) Interferon-γ; (B) IL-17; (C) IL-4; (D) FoxP3. * indicates p<0.05.

FIG. 4 shows data concerning nasal p210-CTB immunization induced IL-10-producing CD4⁺ T cells and apoB-100-specific Treg activity in spleen. A) Flow cytometric analysis of cultured spleen cells stained for intracellular IL-10. (B) Representative flow cytometric plots. (C) Splenic effector cells at 2.5×10⁵ cells/well were generated from apoe^(−/−) mice that had been immunized with human apoB-100. The stimulation index represents the ratio of ³Hthymidine uptake upon stimulation with human apoB-100 (20 μg/mL) relative to unstimulated cells. Proliferation of effector cells alone is indicated in the leftmost bar of each group. Addition of purified CD4⁺ T cells from nasally immunized animals is indicated at different ratios to effector cells. * p<0.05.

FIG. 5 shows data concerning a protective effect of nasal p210-CTB immunization on atherosclerotic lesion size does not depend on TGF-β signaling in T cells. (A) Lesion size in the aortic root of apoe^(−/−)×CD4dnTGFβRIItg mice immunized with p210-CTB (black dots) or OVA-CTB (grey dots). Effect of immunization on p210-specific antibody titers of IgG class (B) and IgM (C). * p<0.05.

FIG. 6 shows data concerning lesion size in immunized mice. Fractional area of lesions in aortic root of apoe^(−/−) mice treated nasally with p210-CTB (black bar), OVA-CTB (grey bar) and untreated controls (white bar). Mean+SD values are shown. * indicates p<0.05.

FIG. 7 shows data concerning plasma lipoprotein profiles analyzed by FPLC. Thick lines represent immunized groups: black=p210-CTB, grey=OVA-CTB and white shows the control group. Thin lines represent SEM. CR/VLDL=chylomicrons/very low density lipoproteins; LDL=low density lipoproteins; HDL=high density lipoproteins.

FIG. 8 shows data concerning T cells in lesions in immunized mice, and in particular representative immunohistochemical stains of atherosclerotic lesion in the aortic root of apoe^(−/−) mice. Arrows indicate FoxP3+ cells; they were CD4+ in directly adjacent sections.

FIG. 9 shows data concerning serum IgG1 antibody titers to apoB-100 peptide. ELISA analysis of sera from apoe^(−/−) mice treated with p210-CTB (black bar), OVA-CTB (grey bar) or controls (white bar). * indicates p<0.05.

FIG. 10 shows data concerning serum IgG2a antibody titers to apoB-100 peptide. Apoe^(−/−) mice treated with p210-CTB (black bar), OVA-CTB (grey bar) or controls (white bar). * indicates p<0.05.

FIG. 11 shows data concerning total IgG in sera of immunized mice. Apoe^(−/−) mice treated with p210-CTB (black bar), OVA-CTB (grey bar) or controls (white bar).

FIG. 12 shows data concerning total IgM in sera of immunized mice. Apoe^(−/−) mice treated with p210-CTB (black bar), OVA-CTB (grey bar) or controls (white bar).

FIG. 13 shows data concerning T cell subsets in spleen after immunization. Flow cytometric analysis of intracellular cytokine expression is shown as percentage of cytokine-producing cells per CD4 T cells for each of the three groups. (A) Interferon-γ; (B) IL-17; (C) IL-4; (D) FoxP3.

FIG. 14 shows data concerning abrogation of Treg suppressor effect upon separation of cells. Cocultures of effector cells and CD4⁺ T cells from nasally immunized mice are indicated by plain bars (generated from black=p210-CTB, grey=OVA-CTB and white=control group animals). Abrogated effect of CD4+ T cell-mediated inhibition of proliferation when effector cells and CD4⁺ T cells are cultured separate from each other in a transwell plate (contact inhibition assay).

FIG. 15 shows data concerning cytokine levels for TGF-β and IL-10 in the supernatants from the coculture assay. Striped bars represent effector cells alone. Cocultures of effector cells and CD4⁺ T cells from nasally immunized mice are indicated by plain bars (generated from black=p210-CTB, grey=OVA-CTB and white=control group animals).

FIG. 16 shows data concerning lesion size in immunized mice with defective TGFβ receptors on T cells. Fractional area of the lesion in the aortic root of apoe^(−/−)×CD4dnTGFβRIItg mice treated nasally with p210-CTB (black bar) or OVA-CTB (grey bar). Mean±SD values are shown. * indicates p<0.05.

FIG. 17 shows data concerning mRNA levels in aorta of mice with defective TGFβ receptors on T cells. Real-time reverse-transcription PCR normalized to HPRT in thoracic aorta of apoe^(−/−)×CD4dnTGFβRIItg mice. Of note, the development of functional Foxp3 Treg cells depends on TGFβ. Therefore, detailed information concerning the relevance of FoxP3-expressing cells in apoe^(−/−)×CD4dnTGFβRIItg mice require further investigation.

FIG. 18 shows data concerning IgG antibodies to mouse LDL in mice immunized with LDL or OVA. C57BL/6J mice were immunized with mouse LDL (mLDL) or ovalbumin (OVA). ELISA was performed with serum dilutions in wells coated with mouse LDL, followed by alkaline phosphatase-labeled anti-mouse-IgG. SC, subcutaneous administration, IN, intranasal administration.

DETAILED DESCRIPTION

Provided herein are fusion proteins, compositions, methods and systems that in several embodiments are suitable to be used for immunoprotection against atherosclerosis.

The term “fusion protein” as used herein indicates a protein created through the attaching of two or more polypeptides which originated from separate proteins. In particular fusion proteins can be created by recombinant DNA technology and are typically used in biological research or therapeutics. Fusion proteins can also be created through chemical covalent conjugation with or without a linker between the polypeptides portion of the fusion proteins.

The term “attach” or “attached” as used herein, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment such that for example where a first polypeptide is directly bound to a second polypeptide or material, and the embodiments wherein one or more intermediate compounds, and in particular polypeptides, are disposed between the first polypeptide and the second polypeptide or material.

The term “protein” or “polypeptide” as used herein indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called an oligopeptide. As used herein the terms “amino acid”, “amino acidic monomer”, or “amino acid residue” refer to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers. The term “amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, isotope, or with a different functional group but is otherwise identical to its natural amino acid analog.

In the particular in several embodiments, fusion proteins, compositions methods and systems are described that in several embodiments are suitable for eliciting an antigen-specific T regulatory cells response in an individual.

The term “antigen”, as it is used herein, relates to any substance that, when introduced into the body can stimulate an immune response. Antigens comprise exogenous antigens (antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection) and endogenous antigens or autoantigens (antigens that have been generated within the body). In particular, an “autoantigen” is an antigen that despite being a normal tissue constituent is the target of a humoral or cell-mediated immune response. Exemplary autoantigens comprise autoantigens associated to atherogenesis and/or atherosclerosis provided by low-density lipoprotein and its constituent protein, apoB-100.

The term “regulatory T cell,” “T regulatory cell” or “Treg” as used herein indicates a component of the immune system that suppress immune responses of other cells, and comprises T cells that express the CD8 transmembrane glycoprotein (CD8+ T cells); T cells that express CD4, CD25, and Foxp3 (CD4+CD25+ regulatory T cells); and other T cell types that have suppressive function identifiable by a skilled person. Treg comprise both naturally occurring T cells and T cells generated in vitro.

The term “antigen-specific” as used indicates an immunity response, and in particular, immunological tolerance, for a certain antigen which is characterized by a substantially less or no immune response (and in particular, immunological tolerance) for another antigen. Accordingly, an antigen-specific regulatory T cell, specific for one or more autoantigens is able, under appropriate conditions to minimize to the specific immune response to the one or more autoantigens with substantially less or no minimizing effect on the immune response towards other antigens or autoantigens.

Fusion proteins comprising autoantigen associated with atherogenesis and/or atherosclerosis and related methods and systems are herein described that are capable of eliciting an autoantigen-specific Treg response and that in several embodiments can be used for treating and/or preventing atherosclerosis or a condition associated thereto in an individual.

The term “atherosclerosis” as used herein indicates an inflammatory condition, and in particular the condition in which an artery wall thickens as the result of a build-up of fatty materials such as cholesterol. In some cases, atherosclerosis is treated with statin therapy (1). In several cases, atherosclerosis is a syndrome affecting arterial blood vessels, a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by low-density lipoproteins (LDL, plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high-density lipoproteins (HDL), (see apoA-1 Milano). Lipid retention and modification in the arterial intima in some cases elicit a chronic inflammatory process with autoimmune responses and the development of atherosclerotic lesions (2). Both adaptive and innate immune mechanisms have been described as contributors to this process (3-6). While pattern recognition receptors of innate immunity are believed to account for cholesterol uptake and contribute to activation of macrophages and endothelial cells, antigen-specific T cells recognizing LDL particles in the intima provide strong proinflammatory stimuli that accelerate atherogenesis. Atherosclerosis is commonly referred to as a hardening or furring of the arteries. It is believed to be caused by the formation of multiple plaques within the arteries. Typically, autoimmune responses to LDL contribute to its progression, while immunization with LDL may induce atheroprotective or proatherogenic responses.

The term “treating” or “treatment” as used herein indicates any activity that is part of a medical care for, or that deals with, a condition medically or surgically. The term “preventing” or “prevention” as used herein indicates any activity, which reduces the burden of mortality or morbidity from a condition in an individual. This takes place at primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications.

The term “condition” as used herein indicates as usually the physical status of the body of an individual (as a whole or of one or more of its parts) that does not conform to a physical status of the individual (as a whole or of one or more of its parts) that is associated with a state of complete physical, mental and possibly social well-being. Conditions herein described include but are not limited to disorders and diseases wherein the term “disorder” indicates a condition of the living individual that is associated to a functional abnormality of the body or of any of its parts, and the term “disease” indicates a condition of the living individual that impairs normal functioning of the body or of any of its parts and is typically manifested by distinguishing signs and symptoms. Exemplary conditions include but are not limited to injuries, disabilities, disorders (including mental and physical disorders), syndromes, infections, deviant behaviors of the individual and atypical variations of structure and functions of the body of an individual or parts thereof.

The wording “associated to” as used herein with reference to two items indicates a relation between the two items such that the occurrence of a first item is accompanied by the occurrence of the second item, which includes but is not limited to a cause-effect relation and sign/symptoms-disease relation.

The term “individuals” as used herein indicates a single biological organism such as higher animals and in particular vertebrates such as mammals and more particularly human beings.

In several embodiments, induction of an antigen-specific Tregulatory cell response is provided by a fusion protein comprising an antigenic fragment of apoB-100 and a protein carrier attached directly or through a linker in an fragment:carrier 1:1 proportion.

The term “fragment” as used herein indicates a portion of a polypeptide of any length. A skilled person will understand that the term encompasses peptides of any origin which have a sequence corresponding to the portion of the polypeptide at issue. An antigenic fragment of apoB-100 is accordingly a portion of apoB-100 that presents antigenic properties. Antigenic fragments of apoB-100 herein described also include possible derivatives thereof.

The term “derivative” as used herein with reference to a first polypeptide (e.g., apoB-100 antigenic fragment), indicates a second polypeptide that is structurally related to the first polypeptide and is derivable from the first polypeptide by a modification that introduces a feature that is not present in the first polypeptide, while retaining functional properties of the first polypeptide. Accordingly, a derivative polypeptide of an antigenic fragment of apoB-100, usually differs from the original polypeptide or portion thereof by modification of the amino acidic sequence that might or might not be associated with an additional function not present in the original polypeptide or portion thereof. A derivative polypeptide of an antigenic fragment of apoB-100 retains however antigenic properties comparable to the ones described in connection with apoB-100 or the antigenic fragment thereof. Retaining of one or more antigenic properties can be verified with methods identifiable by a skilled person upon reading of the present disclosure, on the basis of the specific antigenic property of the fragment at issue. Exemplary methods comprise immunizing an animal (e.g. mouse) with a candidate derivative, determining production of antibody specific for the derivative in the animal (e.g. by ELISA such as immunometric ELISA) and comparing the determined antibody production for the candidate derivative with a corresponding antibody production of the fragment. Additional methods to determine further antigenic properties can be identified by a skilled person upon reading of the present disclosure.

The term “protein carrier” as used herein indicates proteins that transport a specific substance or group of substances through intracellular compartments or in extracellular fluids (e.g. in the blood) or else across the cell membrane. Exemplary carrier proteins comprise subunit B of cholera toxin, avidin, BTG protein, bovine G globulin, bovine immunoglobulin G, bovine thyroglobulin, bovine serum albumin (BSA), conalbumin, edestin, exoprotein A from Pseudomonas aeruginosa, hemocyanin from crab Paralithodes camtschatica (HC), Helix promatia haemocyanin (HPH), human serum albumin (HSA), Kunitz trypsin inhibitor from soybeans (KTI), keyhole limpet hemocyanin (KLH), Limulus polyphemus hemocyanin (LPH), ovalbumin, Pam3Cys-Th, polylysine, porcine thyroglobulin (PTG), purified protein derivative (PPD), rabbit serum albumin (RSA), soybean trypsin inhibitor (STI), sunflower globulin (SFG), and additional molecules identifiable by a skilled person. Additional carriers comprise molecule having immunogenic activities including cytokines such as IL-10, IL12, IL-4, IL-16 and transforming growth factor beta (TGFβ).

In some embodiments, attachment of the carrier is performed at the C-terminus or N-terminus of the fragment. In an embodiment the fusion protein can be provided as a single polypeptide through recombinant DNA technology and related processes, such as cloning, chimeric constructs, polymerase chain reaction, and additional procedures identifiable by a skilled person. In some embodiments, attachment can be performed through chemical linkage of the fragment to the carrier using methods also identifiable by a skilled person.

In some embodiments, the antigenic fragment of apoB-100 comprises amino acids 3136-3155 of human apoB-100 (p210) and/or additional fragments selected from the peptides illustrated in the Examples section.

In particular in some embodiments the fragment portion of the fusion product can comprise one or more of peptides P1, P2, P11, P25, P32, P45, P74, P102, P129, P143, P148, P154, P162, P210, P219, and P240. More particularly, in some embodiments the fragment portion of the fusion products can comprise one or more of peptides P2, P45, P102 and P210.

In an embodiment, wherein the fragment portion of the fusion protein comprise more than one peptide, the fragment portion can comprise up to 10 peptides in a construct that, at least in some of those embodiments, is expected to have effects analogous to those of cancer or infectious vaccines, such as the ones described in reference 35 herein incorporated by reference in its entirety. As skilled person will be able to identify suitable combination of peptides for a desired immunogenic, preventive and/or therapeutic effect upon reading of the present disclosure.

In some embodiments, the carrier protein can comprise at least one monomer of the subunit B of cholera toxin which can be formed by a recombinant pentameric B oligomer that is capable of binding GM-1 receptors (e.g. on the surface of intestinal epithelial cells). In particular, in some embodiments, the carrier protein can be formed by at least one of five identical monomers with a molecular weight of approximately 11.6 kDa recombinant pentameric B oligomer molecule. In some of those embodiments, the monomers are tightly linked into a trypsin-resistant pentameric ring-like structure with a molecular weight of approximately 58 kDa.

In some embodiments, the antigenic fragments can be attached to the carrier molecule using biological genetic engineering to produce a fusion protein (with single or multiple copies of the immunogenic peptide) and procedures identifiable by a skilled person upon reading of the present disclosure.

In some embodiments, the antigenic fragments can be attached to the carrier molecule using chemical covalent conjugation (with or without a linker group) and procedures identifiable by a skilled person upon reading of the present disclosure.

In some embodiments, fusion products or antigenic fragments can be used in the treatment of atherosclerosis and or for induction of regulatory T-cells

In some embodiments, antigen-specific immunomodulation by vaccination is an approach used to prevent or treat chronic inflammatory diseases associated to atherogenesis. In some of those cases, by mobilizing protective immune responses in an antigen-specific manner, side effects due to hampered host defense against infections can be avoided. Exemplary protocols comprise protocols described to treat atherosclerosis in mice and rabbits immunized with LDL, beta2-glycoprotein-Ib, or heat-shock protein 60/65, and parenteral (7-10) as well as oral (11-14) immunization reduced atherosclerotic disease in hyperlipidemic animals.

In some embodiments, antigen-specific immunoprotection can be achieved through several different mechanisms, such as production of protective antibodies, deletion or inactivation (anergy) of pathogenic T cell clones, or induction of suppressive cellular immunity mediated by the family of regulatory T cells (Treg) (15-16). In some of those embodiments, immunization with immunodominant peptide sequences can be performed in several cases in alternative to immunization with LDL particles (17-18).

In an embodiment, an immunization protocol that facilitates selective targeting of antigen-specific regulatory T cells can be performed. The type of immune response triggered is largely determined by the route of immunization.

In several embodiments, fusion products or antigenic fragments herein described can be administered to an individual using various routes of administration including subcutaneous, intramuscular, parenteral, and systemic and mucosal administration such as oral and/or nasal. In particular, the mucosal linings of airways and intestines contain lymphatic tissue that, when exposed to antigen, elicits anti-inflammatory, immunosuppressive responses (19). Distinct immunological features of the respiratory and intestinal mucosa lead to partly different types of protective immunity upon antigen exposure by the nasal or oral route (20). In some embodiments, the B subunit of cholera toxin (CTB) promotes uptake of antigen via the nasal and oral mucosa and induction of protective immunity (21,22).

In some embodiments, administration of carrier/adjuvant/peptide vaccines is performed for a time and under condition to activate regulatory T cells and down-regulate pathogenic autoimmunity against Apo B.

In particular, in some embodiments, administration of a fusion protein is performed by nasal administration of an apoB-100 peptide-CTB fusion protein (p210-CTB). In some embodiments, treatment with p210-CTB significantly reduced atherosclerosis in apoe^(−/−) mice and was associated with induction of antigen-specific Treg activity.

In some embodiments, intranasal immunization with an apoB-100 fusion protein induces antigen-specific regulatory T cells and reduces atherosclerosis.

In several embodiments, nasal administration of an apoB-100 peptide fused to CTB attenuates atherosclerosis and induces regulatory Tr1 cells that inhibit T effector responses to apolipoprotein B-100.

In some embodiments, fusion products, compositions and/or methods compositions herein described can be used a novel strategy for induction of atheroprotective immunity, involving antigen-specific regulatory T cells. In particular, in several By nasal administration of a fusion protein between an immunodominant peptide of apoB-100 and immunomodulatory CTB, we were able to induce an atheroprotective immune response to apoB-100 that involved expansion of antigen-specific regulatory CD4⁺ T cells and inhibition of aortic lesion development.

In several embodiments, induction of antigen-specific Treg with fusion protein methods and systems herein described provides atheroprotection using parenteral or oral routes for LDL immunization. Additionally, results illustrated in the Examples section concerning induction of antigen-specific atheroprotective immunity mucosal immunization in apoe^(−/−) mice, which spontaneously develop atherosclerosis and are therefore already sensitized to plaque antigens such as LDL particles at the time of vaccination, supports the conclusion that a comparable approach in humans with pre-existing lesions is expected to provide immunization.

In some embodiments, described fusion proteins trigger a mechanism of atheroprotection where the atheroprotective effect paralleled an induction of Treg suppression of apoB-100-specific effector T cells and an increase in IL-10⁺ CD4⁺ T cells. In particular, in some embodiments, nasal immunization with p210-CTB protects against atherosclerosis by induction of antigen-specific, IL-10⁺ regulatory Tr1 cells. A possible explanation that is provided herein for guidance purpose only and it is not intended to be limiting is that atheroprotection in several cases does not involve the immunosuppressive cytokine TGF-β since nasal immunization with p210-CTB reduced atherosclerosis also in mice lacking functional TGF-β receptors on T cells.

In some embodiments, fusion protein herein described provide an antigen-specific as well as antigen-independent effects similar to what reported in studies of Treg (25). In particular, in some embodiments, Treg suppress conventional effector T cells with the same antigen-specificity. In some embodiments, Treg exert major effects on antigen-presenting cells in an antigen-independent manner. In some embodiments, the antigen-specific atheroprotection is paralleled by inhibition of apoB100-specific effector T cells by Treg specific for p210 but not OVA. These findings support a protective role for autoantigen-specific Treg in atherosclerosis.

In some embodiments, two major types of Treg induced in the periphery by antigen exposure have been identified: FoxP3⁺ induced Treg (Th3) (14) and Tr1 cells (26). Tr1 cells are FoxP3 negative, secrete IL-10, and are believed to play an important role when regulatory immunity is induced by nasal immunization (27,28). In some embodiments, where atheroprotection is induced by nasal immunization and associated with suppressor T cell activity and IL-10 producing CD4+ T cells, administration of fusion protein herein described is associated with Tr1 induction by p210-CTB. CD4+ T cells with antigen-specific suppressor activity were derived from spleen, a known reservoir of Tr1 cells (26).

In some embodiments, FoxP3⁺ Treg can contribute to atheroprotection in this model following administration of a fusion protein herein described as indicated by an increase of FoxP3 mRNA was increased in the aorta of nasally immunized mice. In some embodiments, these cells can not only act directly to control proinflammatory effector T cells but also promote the activation of Tr1 cells (19). In some embodiments, wherein abrogation of TGF-β signaling is detected, Tr1 cells do not extinguish atheroprotection.

In some embodiments, where Treg markers are elevated also in OVA-CTB immunized mice, antigenically nonspecific effects can synergize with antigen-specific ones to confer protection.

In some embodiments, antibodies to the apoB-100 peptide sequence are induced by nasal immunization, but do not crossreact with native mouse LDL particles. Furthermore, in some embodiments where particular antibody titers are not correlated with lesion size and no difference in lipoprotein profiles is detected between apoB-100-CTB immunized and OVA-CTB, immunized mice atheroprotection is associated to immunomodulation rather than antibody-dependent elimination of LDL.

In some embodiments, fusion proteins herein described are comprised in a composition together with suitable adjuvant and/or excipients.

The term adjuvant as used herein indicates a pharmacological or immunological agent that modify the effect of other agents (e.g., drugs, vaccines) while having few if any direct effects when given by themselves. They are often included in vaccines to enhance the recipient's immune response to a supplied antigen while keeping the injected foreign material at a minimum. Types of adjuvants include: Immunologic adjuvant that stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself.

The term excipients as used herein indicates an inactive substance used as a carrier for the active ingredients of a medication. Exemplary excipients can also be used to bulk up formulations that contain very potent active ingredients, to allow for convenient and accurate dosage. In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of the active substance concerned. Depending on the route of administration, and form of medication, different excipients may be used that are identifiable by a skilled person.

In some embodiments, the compositions comprises selected (immunogenic) peptide fragments of apoB-100 (single or multiple copies) fused with a carrier molecule and possibly toxins/toxoids: tetanus toxoid, diphtheria toxoid, B subunit of cholera toxin, as well as BSA, HSA, rHSA, KLH, ovalbumin,

In some embodiments, the adjuvants and excipients are pharmaceutically acceptable and the resulting composition is a pharmaceutical composition. In some of those embodiments, the pharmaceutical composition is a vaccine.

In some embodiments, adjuvants are components of the vaccine formulation that enhance immunogenicity of the antigen, for instance by promoting their uptake by antigen-presenting cells (17,29). Interestingly, two studies documented an atheroprotective effect of complete Freund's adjuvant in hypercholesterolemic ldlr^(−/−) and apoe^(−/−) mice (30-31). In a recent study, subcutaneous administration of alum adjuvant was shown to increase antigen uptake and activation of cellular immune responses in hypercholesterolemic mice (32). In some embodiments, a specific antibody response against the apoB-100 peptide and an immunomodulatory cytokine profile in aortas of mice immunized with OVA-CTB described herein is in line with such an adjuvant effect. This further underlines the importance of using optimal immunomodulatory components in vaccine preparations.

In several embodiments, atheroprotective vaccine is provided by targeting a peptide of the LDL protein constituent apolipoprotein B-100 to the nasal mucosa to induce a protective mucosal immune response.

Further details concerning the implementation of the fusion products methods herein described including systems for performance of the methods which can be in the form of kit of parts as well as related compositions including donors, acceptors, compounds and other reagents together with suitable carrier, agent or auxiliary agent of the compositions, and generally manufacturing and packaging of the kit, can be identified by the person skilled in the art upon reading of the present disclosure.

EXAMPLES

The fusion proteins and related compositions methods and systems herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate an exemplary immunization performed with a fusion proteins comprising amino acids 3136-3155 of human apoB-100 (P210) fused with CTB identified also as P210-CTB. A person skilled in the art will appreciate the applicability of the features described in detail for CTB-P210 for additional fusion protein comprising another antigenic peptide of apoB-100 and a carrier molecule according to the present disclosure.

More particular, in the following examples the recombinant proteins are made from the desired amino acid sequence of human apoB-100 fused with a carrier amino acid sequence.

Antigen-specific Treg activity was analyzed in the following way: apoe^(−/−) mice were immunized subcutaneously with apoB-100 to generate effector T cells. CD4⁺ T cells from these mice were exposed to antigen and activation recorded as DNA synthesis. CD4⁺ T cells from apoe^(−/−) mice immunized intranasally with p210-CTB were added to effector T cell preparations and Treg activity was recorded as inhibition of DNA synthesis. Intracellular staining was performed on CD4⁺ T cells to characterize cytokine production and T cell subtype.

Additional details concerning procedures used and results obtained are reported below.

Example 1 Generation of Peptide Library

Specific immunogenic epitopes by focusing on the single protein found in LDL, apolipoprotein B-100 (apoB-100) were characterized. A peptide library comprised of 302 peptides, 20 amino acid residues in length, covering the complete 4563 amino acid sequence of human apoB-100 was produced. The peptides were produced with a 5 amino acid overlap to cover all sequences at break points. Peptides were numbered 1-302 starting at the N-terminal of apoB-100 as indicated in Table 1 below.

TABLE 1 Peptide Sequence ApoB-100 aa SEQ ID NO P1: EEEML ENVSL VCPKD ATRFK aa 1-20 SEQ ID NO: 1 P2: ATRFK HLRKY TYNYE AESSS aa 16-35 SEQ ID NO: 2 P3: AESSS GVPGT ADSRS ATRIN aa 31-50 SEQ ID NO: 3 P4: ATRIN CKVEL EVPQL CSFIL aa 46-65 SEQ ID NO: 4 P5: CSFIL KTSQC TLKEV YGFNP aa 61-80 SEQ ID NO: 5 P6: YGFNP EGKAL LKKTK NSEEF aa 76-95 SEQ ID NO: 6 P7: NSEEF AAAMS RYELK LAIPE aa 91-110 SEQ ID NO: 7 P8: LAIPE GKQVF LYPEK DEPTY aa 106-125 SEQ ID NO: 8 P9: DEPTY ILNIK RGIIS ALLVP aa 121-140 SEQ ID NO: 9 P10: ALLVP PETEE AKQVL FLDTV aa 136-155 SEQ ID NO: 10 P11: FLDTV YGNCS THFTV KTRKG aa 151-170 SEQ ID NO: 11 P12: KTRKG NVATE ISTER DLGQC aa 166-185 SEQ ID NO: 12 P13: DLGQC DRFKP IRTGI SPLAL aa 181-200 SEQ ID NO: 13 P14: SPLAL IKGMT RPLST LISSS aa 196-215 SEQ ID NO: 14 P15: LISSS QSCQY TLDAK RKHVA aa 211-230 SEQ ID NO: 15 P16: RKHVA EAICK EQHLF LPFSY aa 226-245 SEQ ID NO: 16 P17: LPFSY NNKYG MVAQV TQTLK aa 241-260 SEQ ID NO: 17 P18: TQTLK LEDTP KINSR FFGEG aa 256-275 SEQ ID NO: 18 P19: FFGEG TKKMG LAFES TKSTS aa 271-290 SEQ ID NO: 19 P20: TKSTS PPKQA EAVLK TLQEL aa 286-305 SEQ ID NO: 20 P21: TLQEL KKLTI SEQNI QRANL aa 301-320 SEQ ID NO: 21 P22: QRANL FNKLV TELRG LSDEA aa 316-335 SEQ ID NO: 22 P23: LSDEA VTSLL PQLIE VSSPI aa 331-350 SEQ ID NO: 23 P24: VSSPI TLQAL VQCGQ PQCST aa 346-365 SEQ ID NO: 24 P25: PQCST HILQW LKRVH ANPLL aa 361-380 SEQ ID NO: 25 P26: ANPLL IDVVT YLVAL IPEPS aa 376-395 SEQ ID NO: 26 P27: IPEPS AQQLR EIFNM ARDQR aa 391-410 SEQ ID NO: 27 P28: ARDQR SRATL YALSH AVNNY aa 406-425 SEQ ID NO: 28 P29: AVNNY HKTNP TGTQE LLDIA aa 421-440 SEQ ID NO: 29 P30: LLDIA NYLME QIQDD CTGDE aa 436-455 SEQ ID NO: 30 P31: CTGDE DYTYL ILRVI GNMGQ aa 451-470 SEQ ID NO: 31 P32: GNMGQ TMEQL TPELK SSILK aa 466-485 SEQ ID NO: 32 P33: SSILK CVQST KPSLM IQKAA aa 481-500 SEQ ID NO: 33 P34: IQKAA IQALR KMEPK DKDQE aa 496-515 SEQ ID NO: 34 P35: DKDQE VLLQT FLDDA SPGDK aa 511-530 SEQ ID NO: 35 P36: SPGDK RLAAY LMLMR SPSQA aa 526-545 SEQ ID NO: 36 P37: SPSQA DINKI VQILP WEQNE aa 541-560 SEQ ID NO: 37 P38: WEQNE QVKNF VASHI ANILN aa 556-575 SEQ ID NO: 38 P39: ANILN SEELD IQDLK KLVKE aa 571-590 SEQ ID NO: 39 P40: KLVKE ALKES QLPTV MDFRK aa 586-605 SEQ ID NO: 40 P41: MDFRK FSRNY QLYKS VSLPS aa 601-620 SEQ ID NO: 41 P42: VSLPS LDPAS AKIEG NLIFD aa 616-635 SEQ ID NO: 42 P43: NLIFD PNNYL PKESM LKTTL aa 631-650 SEQ ID NO: 43 P44: LKTTL TAFGF ASADL IEIGL aa 646-665 SEQ ID NO: 44 P45: IEIGL EGKGF EPTLE ALFGK aa 661-680 SEQ ID NO: 45 P46: ALFGK QGFFP DSVNK ALYWV aa 676-695 SEQ ID NO: 46 P47: ALYWV NGQVP DGVSK VLVDH aa 691-710 SEQ ID NO: 47 P48: VLVDH FGYTK DDKHE QDMVN aa 706-725 SEQ ID NO: 48 P49: QDMVN GIMLS VEKLI KDLKS aa 721-740 SEQ ID NO: 49 P50: KDLKS KEVPE ARAYL RILGE aa 736-755 SEQ ID NO: 50 P51: RILGE ELGFA SLHDL QLLGK aa 751-770 SEQ ID NO: 51 P52: QLLGK LLLMG ARTLQ GIPQM aa 766-785 SEQ ID NO: 52 P53: GIPQM IGEVI RKGSK NDFFL aa 781-800 SEQ ID NO: 53 P54: NDFFL HYIFM ENAFE LPTGA aa 796-815 SEQ ID NO: 54 P55: LPTGA GLQLQ ISSSG VIAPG aa 811-830 SEQ ID NO: 55 P56: VIAPG AKAGV KLEVA NMQAE aa 826-845 SEQ ID NO: 56 P57: NMQAE LVAKP SVSVE FVTNM aa 841-860 SEQ ID NO: 57 P58: FVTNM GIIIP DFARS GVQMN aa 856-875 SEQ ID NO: 58 P59: GVQMN TNFFH ESGLE AHVAL aa 871-890 SEQ ID NO: 59 P60: AHVAL KAGKL KFIIP SPKRP aa 886-905 SEQ ID NO: 60 P61: SPKRP VKLLS GGNTL HLVST aa 901-920 SEQ ID NO: 61 P62: HLVST TKTEV IPPLI ENRQS aa 916-935 SEQ ID NO: 62 P63: ENRQS WSVCK QVFPG LNYCT aa 931-950 SEQ ID NO: 63 P64: LNYCT SGAYS NASST DSASY aa 946-965 SEQ ID NO: 64 P65: DSASY YPLTG DTRLE LELRP aa 961-980 SEQ ID NO: 65 P66: LELRP TGEIE QYSVS ATYEL aa 976-995 SEQ ID NO: 66 P67: ATYEL QREDR ALVDT LKFVT aa 991-1010 SEQ ID NO: 67 P68: LKFVT QAEGA KQTEA TMTFK aa 1006-1025 SEQ ID NO: 68 P69: TMTFK YNRQS MTLSS EVQIP aa 1021-1040 SEQ ID NO: 69 P70: EVQIP DFDVD LGTIL RVNDE aa 1036-1055 SEQ ID NO: 70 P71: RVNDE STEGK TSYRL TLDIQ aa 1051-1070 SEQ ID NO: 71 P72: TLDIQ NKKIT EVALM GHLSC aa 1066-1085 SEQ ID NO: 72 P73: GHLSC DTKEE RKIKG VISIP aa 1081-1100 SEQ ID NO: 73 P74: VISIP RLQAE ARSEI LAHWS aa 1096-1115 SEQ ID NO: 74 P75: LAHWS PAKLL LQMDS SATAY aa 1111-1130 SEQ ID NO: 75 P76: SATAY GSTVS KRVAW HYDEE aa 1126-1145 SEQ ID NO: 76 P77: HYDEE KIEFE WNTGT NVDTK aa 1141-1160 SEQ ID NO: 77 P78: NVDTK KMTSN FPVDL SDYPK aa 1156-1175 SEQ ID NO: 78 P79: SDYPK SLHMY ANRLL DHRVP aa 1171-1190 SEQ ID NO: 79 P80: DHRVP ETDMT FRHVG SKLIV aa 1186-1205 SEQ ID NO: 80 P81: SKLIV AMSSW LQKAS GSLPY aa 1201-1220 SEQ ID NO: 81 P82: GSLPY TQTLQ DHLNS LKEFN aa 1216-1235 SEQ ID NO: 82 P83: LKEFN LQNMG LPDFH IPENL aa 1231-1250 SEQ ID NO: 83 P84: IPENL FLKSD GRVKY TLNKN aa 1246-1260 SEQ ID NO: 84 P85: TLNKN SLKIE IPLPF GGKSS aa 1261-1280 SEQ ID NO: 85 P86: GGKSS RDLKM LETVR TPALH aa 1276-1295 SEQ ID NO: 86 P87: TPALH FKSVG FHLPS REFQV aa 1291-1310 SEQ ID NO: 87 P88: REFQV PTFTI PKLYQ LQVPL aa 1306-1325 SEQ ID NO: 88 P89: LQVPL LGVLD LSTNV YSNLY aa 1321-1340 SEQ ID NO: 89 P90: YSNLY NWSAS YSGGN TSTDH aa 1336-1355 SEQ ID NO: 90 P91: TSTDH FSLRA RYHMK ADSVV aa 1351-1370 SEQ ID NO: 91 P92: ADSVV DLLSY NVQGS GETTY  aa 1366-1385 SEQ ID NO: 92 P93: GETTY DHKNT FTLSC DGSLR aa 1381-1400 SEQ ID NO: 93 P94: DGSLR HKFLD SNIKF SHVEK aa 1396-1415 SEQ ID NO: 94 P95: SHVEK LGNNP VSKGL LIFDA aa 1411-1430 SEQ ID NO: 95 P96: LIFDA SSSWG PQMSA SVHLD aa 1426-1445 SEQ ID NO: 96 P97: SVHLD SKKKQ HLFVK EVKID aa 1441-1460 SEQ ID NO: 97 P98: EVKID GQFRV SSFYA KGTYG aa 1456-1475 SEQ ID NO: 98 P99: KGTYG LSCQR DPNTG RLNGE aa 1471-1490 SEQ ID NO: 99 P100: RLNGE SNLRF NSSYL QGTNQ aa 1486-1505 SEQ ID NO: 100 P101: QGTNQ ITGRY EDGTL SLTST aa 1501-1520 SEQ ID NO: 101 P102: SLTST SDLQS GIIKN TASLK aa 1516-1535 SEQ ID NO: 102 P103: TASLK YENYE LTLKS DTNGK aa 1531-1550 SEQ ID NO: 103 P104: DTNGK YKNFA TSNKM DMTFS aa 1546-1565 SEQ ID NO: 104 P105: DMTFS KQNAL LRSEY QADYE aa 1561-1580 SEQ ID NO: 105 P106: QADYE SLRFF SLLSG SLNSH aa 1576-1595 SEQ ID NO: 106 P107: SLNSH GLELN ADILG TDKIN aa 1591-1610 SEQ ID NO: 107 P108: TDKIN SGAHK ATLRI GQDGI aa 1606-1625 SEQ ID NO: 108 P109: GQDGI STSAT TNLKC SLLVL aa 1621-1640 SEQ ID NO: 109 P110: SLLVL ENELN AELGL SGASM aa 1636-1655 SEQ ID NO: 110 P111: SGASM KLTTN GRFRE HNAKF aa 1651-1670 SEQ ID NO: 111 P112: HNAKF SLDGK AALTE LSLGS aa 1666-1685 SEQ ID NO: 112 P113: LSLGS AYQAM ILGVD SKNIF aa 1681-1700 SEQ ID NO: 113 P114: SKNIF NFKVS QEGLK LSNDM aa 1696-1715 SEQ ID NO: 114 P115: LSNDM MGSYA EMKFD HTNSL aa 1711-1730 SEQ ID NO: 115 P116: HTNSL NIAGL SLDFS SKLDN aa 1726-1745 SEQ ID NO: 116 P117: SKLDN IYSSD KFYKQ TVNLQ aa 1741-1760 SEQ ID NO: 117 P118: TVNLQ LQPYS LVTTL NSDLK aa 1756-1775 SEQ ID NO: 118 P119: NSDLK YNALD LTNNG KLRLE aa 1771-1790 SEQ ID NO: 119 P120: KLRLE PLKLH VAGNL KGAYQ aa 1786-1805 SEQ ID NO: 120 P121: KGAYQ NNEIK HIYAI SSAAL aa 1801-1820 SEQ ID NO: 121 P122: SSAAL SASYK ADTVA KVQGV aa 1816-1835 SEQ ID NO: 122 P123: KVQGV EFSHR LNTDI AGLAS aa 1831-1850 SEQ ID NO: 123 P124: AGLAS AIDMS TNYNS DSLHF aa 1846-1865 SEQ ID NO: 124 P125: DSLHF SNVFR SVMAP FTMTI aa 1861-1880 SEQ ID NO: 125 P126: FTMTI DAHTN GNGKL ALWGE aa 1876-1895 SEQ ID NO: 126 P127: ALWGE HTGQL YSKFL LKAEP aa 1891-1910 SEQ ID NO: 127 P128: LKAEP LAFTF SHDYK GSTSH aa 1906-1925 SEQ ID NO: 128 P129: GSTSH HLVSR KSISA ALEHK aa 1921-1940 SEQ ID NO: 129 P130: ALEHK VSALL TPAEQ TGTWK aa 1936-1955 SEQ ID NO: 130 P131: TGTWK LKTQF NNNEY SQDLD aa 1951-1970 SEQ ID NO: 131 P132: SQDLD AYNTK DKIGV ELTGR aa 1966-1985 SEQ ID NO: 132 P133: ELTGR TLADL TLLDS PIKVP aa 1981-2000 SEQ ID NO: 133 P134: PIKVP LLLSE PINII DALEM aa 1996-2015 SEQ ID NO: 134 P135: DALEM RDAVE KPQEF TIVAF aa 2011-2030 SEQ ID NO: 135 P136: TIVAF VKYDK NQDVH SINLP aa 2026-2045 SEQ ID NO: 136 P137: SINLP FFETL QEYFE RNRQT aa 2041-2060 SEQ ID NO: 137 P138: RNRQT IIVVV ENVQR NLKHI aa 2056-2075 SEQ ID NO: 138 P139: NLKHI NIDQF VRKYR AALGK aa 2071-2090 SEQ ID NO: 139 P140: AALGK LPQQA NDYLN SFNWE aa 2086-2105 SEQ ID NO: 140 P141: SFNWE RQVSH AKEKL TALTK aa 2101-2120 SEQ ID NO: 141 P142: TALTK KYRIT ENDIQ IALDD aa 2116-2135 SEQ ID NO: 142 P143: IALDD AKINF NEKLS QLQTY aa 2131-2150 SEQ ID NO: 143 P144: QLQTY MIQFD QYIKD SYDLH aa 2146-2165 SEQ ID NO: 144 P145: SYDLH DLKIA IANII DEIIE aa 2161-2180 SEQ ID NO: 145 P146: DEIIE KLKSL DEHYH IRVNL aa 2176-2195 SEQ ID NO: 146 P147: IRVNL VKTIH DLHLF IENID aa 2191-2210 SEQ ID NO: 147 P148: IENID FNKSG SSTAS WIQNV aa 2206-2225 SEQ ID NO: 148 P149: WIQNV DTKYQ IRIQI QEKLQ aa 2221-2240 SEQ ID NO: 149 P150: QEKLQ QLKRH IQNID IQHLA aa 2236-2255 SEQ ID NO: 150 P151: IQHLA GKLKQ HIEAI DVRVL aa 2251-2270 SEQ ID NO: 151 P152: DVRVL LDQLG TTISF ERIND aa 2266-2285 SEQ ID NO: 152 P153: ERIND VLEHV KHFVI NLIGD aa 2281-2300 SEQ ID NO: 153 P154: NLIGD FEVAE KINAF RAKVH aa 2296-2315 SEQ ID NO: 154 P155: RAKVH ELIER YEVDQ QIQVL aa 2311-2330 SEQ ID NO: 155 P156: QIQVL MDKLV ELTHQ YKLKE aa 2326-2345 SEQ ID NO: 156 P157: YKLKE TIQKL SNVLQ QVKIK aa 2341-2360 SEQ ID NO: 157 P158: QVKIK DYFEK LVGFI DDAVK aa 2356-2375 SEQ ID NO: 158 P159: DDAVK KLNEL SFKTF IEDVN aa 2371-2390 SEQ ID NO: 159 P160: IEDVN KFLDM LIKKL KSFDY aa 2386-2405 SEQ ID NO: 160 P161: KSFDY HQFVD ETNDK IREVT aa 2401-2420 SEQ ID NO: 161 P162: IREVT QRLNG EIQAL ELPQK aa 2416-2435 SEQ ID NO: 162 P163: ELPQK AEALK LFLEE TKATV aa 2431-2450 SEQ ID NO: 163 P164: TKATV AVYLE SLQDT KITLI aa 2446-2465 SEQ ID NO: 164 P165: KITLI INWLQ EALSS ASLAH aa 2461-2480 SEQ ID NO: 165 P166: ASLAH MKAKF RETLE DTRDR aa 2476-2495 SEQ ID NO: 166 P167: DTRDR MYQMD IQQEL QRYLS aa 2491-2510 SEQ ID NO: 167 P168: QRYLS LVGQV YSTLV TYISD aa 2506-2515 SEQ ID NO: 168 P169: TYISD WWTLA AKNLT DFAEQ aa 2521-2540 SEQ ID NO: 169 P170: DFAEQ YSIQD WAKRM KALVE aa 2536-2555 SEQ ID NO: 170 P171: KALVE QGFTV PEIKT ILGTM aa 2551-2570 SEQ ID NO: 171 P172: ILGTM PAFEV SLQAL QKATF aa 2566-2585 SEQ ID NO: 172 P173: QKATF QTPDF IVPLT DLRIP aa 2581-2600 SEQ ID NO: 173 P174: DLRIP SVQIN FKDLK NIKIP aa 2596-2615 SEQ ID NO: 174 P175: NIKIP SRFST PEFTI LNTFH aa 2611-2630 SEQ ID NO: 175 P176: LNTFH IPSFT IDFVE MKVKI aa 2626-2645 SEQ ID NO: 176 P177: MKVKI IRTID QMQNS ELQWP aa 2641-2660 SEQ ID NO: 177 P178: ELQWP VPDIY LRDLK VEDIP aa 2656-2675 SEQ ID NO: 178 P179: VEDIP LARIT LPDFR LPEIA aa 2671-2690 SEQ ID NO: 179 P180: LPEIA IPEFI IPTLN LNDFQ aa 2686-2705 SEQ ID NO: 180 P181: LNDFQ VPDLH IPEFQ LPHIS aa 2701-2720 SEQ ID NO: 181 P182: LPHIS HTIEV PTFGK LYSIL aa 2716-2735 SEQ ID NO: 182 P183: LYSIL KIQSP LFTLD ANADI aa 2731-2750 SEQ ID NO: 183 P184: ANADI GNGTT SANEA GIAAS aa 2746-2765 SEQ ID NO: 184 P185: GIAAS ITAKG ESKLE VLNFD aa 2761-2780 SEQ ID NO: 185 P186: VLNFD FQANA QLSNP KINPL aa 2776-2795 SEQ ID NO: 186 P187: KINPL ALKES VKFSS KYLRT aa 2791-2810 SEQ ID NO: 187 P188: KYLRT EHGSE MLFFG NAIEG aa 2806-2825 SEQ ID NO: 188 P189: NAIEG KSNTV ASLHT EKNTL aa 2821-2840 SEQ ID NO: 189 P190: EKNTL ELSNG VIVKI NNQLT aa 2836-2855 SEQ ID NO: 190 P191: NNQLT LDSNT KYFHK LNIPK aa 2851-2870 SEQ ID NO: 191 P192: LNIPK LDFSS QADLR NEIKT aa 2866-2885 SEQ ID NO: 192 P193: NEIKT LLKAG HIAWT SSGKG aa 2881-2900 SEQ ID NO: 193 P194: SSGKG SWKWA CPRFS DEGTH aa 2896-2915 SEQ ID NO: 194 P195: DEGTH ESQIS FTIEG PLTSF aa 2911-2930 SEQ ID NO: 195 P196: PLTSF GLSNK INSKH LRVNQ aa 2926-2945 SEQ ID NO: 196 P197: LRVNQ NLVYE SGSLN FSKLE aa 2941-2960 SEQ ID NO: 197 P198: FSKLE IQSQV DSQHV GHSVL aa 2956-2975 SEQ ID NO: 198 P199: GHSVL TAKGM ALFGE GKAEF aa 2971-2990 SEQ ID NO: 199 P200: GKAEF TGRHD AHLNG KVIGT aa 2986-3005 SEQ ID NO: 200 P201: KVIGT LKNSL FFSAQ PFEIT aa 3001-3020 SEQ ID NO: 201 P202: PFEIT ASTNN EGNLK VRFPL aa 3016-3035 SEQ ID NO: 202 P203: VRFPL RLTGK IDFLN NYALF aa 3031-3050 SEQ ID NO: 203 P204: NYALF LSPSA QQASW QVSAR aa 3046-3065 SEQ ID NO: 204 P205: QVSAR FNQYK YNQNF SAGNN aa 3061-3080 SEQ ID NO: 205 P206: SAGNN ENIME AHVGI NGEAN aa 3076-3095 SEQ ID NO: 206 P207: NGEAN LDFLN IPLTI PEMRL aa 3091-3110 SEQ ID NO: 207 P208: PEMRL PYTII TTPPL KDFSL aa 3106-3125 SEQ ID NO: 208 P209: KDFSL WEKTG LKEFL KTTKQ aa 3121-3140 SEQ ID NO: 209 P210: KTTKQ SFDLS VKAQY KKNKH aa 3136-3155 SEQ ID NO: 210 P211: KKNKH RHSIT NPLAV LCEFI aa 3151-3170 SEQ ID NO: 211 P212: LCEFI SQSIK SFDRH FEKNR aa 3166-3185 SEQ ID NO: 212 P213: FEKNR NNALD FVTKS YNETK aa 3181-3200 SEQ ID NO: 213 P214: YNETK IKFDK YKAEK SHDEL aa 3196-3215 SEQ ID NO: 214 P215: SHDEL PRTFQ IPGYT VPVVN aa 3211-3230 SEQ ID NO: 215 P216: VPVVN VEVSP FTIEM SAFGY aa 3226-3245 SEQ ID NO: 216 P217: SAFGY VFPKA VSMPS FSILG aa 3241-3260 SEQ ID NO: 217 P218: FSILG SDVRV PSYTL ILPSL aa 3256-3275 SEQ ID NO: 218 P219: ILPSL ELPVL HVPRN LKLSL aa 3271-3290 SEQ ID NO: 219 P220: LKLSL PHFKE LCTIS HIFIP aa 3286-3305 SEQ ID NO: 220 P221: HIFIP AMGNI TYDFS FKSSV aa 3301-3320 SEQ ID NO: 221 P222: FKSSV ITLNT NAELF NQSDI aa 3316-3335 SEQ ID NO: 222 P223: NQSDI VAHLL SSSSS VIDAL aa 3331-3350 SEQ ID NO: 223 P224: VIDAL QYKLE GTTRL TRKRG aa 3346-3365 SEQ ID NO: 224 P225: TRKRG LKLAT ALSLS NKFVE aa 3361-3380 SEQ ID NO: 225 P226: NKFVE GSHNS TVSLT TKNME aa 3376-3395 SEQ ID NO: 226 P227: TKNME VSVAK TTKAE IPILR aa 3391-3410 SEQ ID NO: 227 P228: IPILR MNFKQ ELNGN TKSKP aa 3406-3425 SEQ ID NO: 228 P229: TKSKP TVSSS MEFKY DFNSS aa 3421-3440 SEQ ID NO: 229 P230: DFNSS MLYST AKGAV DHKLS aa 3436-3455 SEQ ID NO: 230 P231: DHKLS LESLT SYFSI ESSTK aa 3451-3470 SEQ ID NO: 231 P232: ESSTK GDVKG SVLSR EYSGT aa 3466-3485 SEQ ID NO: 232 P233: EYSGT IASEA NTYLN SKSTR aa 3481-3500 SEQ ID NO: 233 P234: SKSTR SSVKL QGTSK IDDIW aa 3496-3515 SEQ ID NO: 234 P235: IDDIW NLEVK ENFAG EATLQ aa 3511-3530 SEQ ID NO: 235 P236: EATLQ RIYSL WEHST KNHLQ aa 3526-3545 SEQ ID NO: 236 P237: KNHLQ LEGLF FTNGE HTSKA aa 3541-3560 SEQ ID NO: 237 P238: HTSKA TLELS PWQMS ALVQV aa 3556-3575 SEQ ID NO: 238 P239: ALVQV HASQP SSFHD FPDLG aa 3571-3590 SEQ ID NO: 239 P240: FPDLG QEVAL NANTK NQKIR aa 3586-3605 SEQ ID NO: 240 P241: NQKIR WKNEV RIHSG SFQSQ aa 3601-3620 SEQ ID NO: 241 P242: SFQSQ VELSN DQEKA HLDIA aa 3616-3635 SEQ ID NO: 242 P243: HLDIA GSLEG HLRFL KNIIL aa 3631-3650 SEQ ID NO: 243 P244: KNIIL PVYDK SLWDF LKLDV aa 3646-3665 SEQ ID NO: 244 P245: LKLDV TTSIG RRQHL RVSTA aa 3661-3680 SEQ ID NO: 245 P246: RVSTA FVYTK NPNGY SFSIP aa 3676-3695 SEQ ID NO: 246 P247: SFSIP VKVLA DKFIT PGLKL aa 3691-3710 SEQ ID NO: 247 P248: PGLKL NDLNS VLVMP TFHVP aa 3706-3725 SEQ ID NO: 248 P249: TFHVP FTDLQ VPSCK LDFRE aa 3721-3740 SEQ ID NO: 249 P250: LDFRE IQIYK KLRTS SFALN aa 3736-3755 SEQ ID NO: 250 P251: SFALN LPTLP EVKFP EVDVL aa 3751-3770 SEQ ID NO: 251 P252: EVDVL TKYSQ PEDSL IPFFE aa 3766-3785 SEQ ID NO: 252 P253: IPFFE ITVPE SQLTV SQFTL aa 3781-3800 SEQ ID NO: 253 P254: SQFTL PKSVS DGIAA LDLNA aa 3796-3815 SEQ ID NO: 254 P255: LDLNA VANKI ADFEL PTIIV aa 3811-3830 SEQ ID NO: 255 P256: PTIIV PEQTI EIPSI KFSVP aa 3826-3845 SEQ ID NO: 256 P257: KFSVP AGIVI PSFQA LTARF aa 3841-3860 SEQ ID NO: 257 P258: LTARF EVDSP VYNAT WSASL aa 3856-3875 SEQ ID NO: 258 P259: WSASL KNKAD YVETV LDSTC aa 3871-3890 SEQ ID NO: 259 P260: LDSTC SSTVQ FLEYE LNVLG aa 3886-3905 SEQ ID NO: 260 P261: LNVLG THKIE DGTLA SKTKG aa 3901-3920 SEQ ID NO: 261 P262: SKTKG TLAHR DFSAE YEEDG aa 3916-3935 SEQ ID NO: 262 P263: YEEDG KFEGL QEWEG KAHLN aa 3931-3950 SEQ ID NO: 263 P264: KAHLN IKSPA FTDLH LRYQK aa 3946-3965 SEQ ID NO: 264 P265: LRYQK DKKGI STSAA SPAVG aa 3961-3980 SEQ ID NO: 265 P266: SPAVG TVGMD MDEDD DFSKW aa 3976-3995 SEQ ID NO: 266 P267: DFSKW NFYYS PQSSP DKKLT aa 3991-4010 SEQ ID NO: 267 P268: DKKLT IFKTE LRVRE SDEET aa 4006-4025 SEQ ID NO: 268 P269: SDEET QIKVN WEEEA ASGLL aa 4021-4040 SEQ ID NO: 269 P270: ASGLL TSLKD NVPKA TGVLY aa 4036-4055 SEQ ID NO: 270 P271: TGVLY DYVNK YHWEH TGLTL aa 4051-4070 SEQ ID NO: 271 P272: TGLTL REVSS KLRRN LQNNA aa 4066-4085 SEQ ID NO: 272 P273: LQNNA EWVYQ GAIRQ IDDID aa 4081-4100 SEQ ID NO: 273 P274: IDDID VRFQK AASGT TGTYQ aa 4096-4115 SEQ ID NO: 274 P275: TGTYQ EWKDK AQNLY QELLT aa 4111-4130 SEQ ID NO: 275 P276: QELLT QEGQA SFQGL KDNVF aa 4126-4145 SEQ ID NO: 276 P277: KDNVF DGLVR VTQKF HMKVK aa 4141-4160 SEQ ID NO: 277 P278: HMKVK HLIDS LIDFL NFPRF aa 4156-4175 SEQ ID NO: 278 P279: NFPRF QFPGK PGIYT REELC aa 4171-4190 SEQ ID NO: 279 P280: REELC TMFIR EVGTV LSQVY aa 4186-4205 SEQ ID NO: 280 P281: LSQVY SKVHN GSEIL FSYFQ aa 4201-4220 SEQ ID NO: 281 P282: FSYFQ DLVIT LPFEL RKHKL aa 4216-4235 SEQ ID NO: 282 P283: RKHKL IDVIS MYREL LKDLS aa 4231-4250 SEQ ID NO: 283 P284: LKDLS KEAQE VFKAI QSLKT aa 4246-4265 SEQ ID NO: 284 P285: QSLKT TEVLR NLQDL LQFIF aa 4261-4280 SEQ ID NO: 285 P286: LQFIF QLIED NIKQL KEMKF aa 4276-4295 SEQ ID NO: 286 P287: KEMKF TYLIN YIQDE INTIF aa 4291-4310 SEQ ID NO: 287 P288: INTIF NDYIP YVFKL LKENL aa 4306-4325 SEQ ID NO: 288 P289: LKENL CLNLH KFNEF IQNEL aa 4321-4340 SEQ ID NO: 289 P290: IQNEL QEASQ ELQQI HQYIM aa 4336-4355 SEQ ID NO: 290 P291: HQYIM ALREE YFDPS IVGWT aa 4351-4370 SEQ ID NO: 291 P292: IVGWT VKYYE LEEKI VSLIK aa 4366-4385 SEQ ID NO: 292 P293: VSLIK NLLVA LKDFH SEYIV aa 4381-4400 SEQ ID NO: 293 P294: SEYIV SASNF TSQLS SQVEQ aa 4396-4415 SEQ ID NO: 294 P295: SQVEQ FLHRN IQEYL SILTD aa 4411-4430 SEQ ID NO: 295 P296: SILTD PDGKG KEKIA ELSAT aa 4426-4445 SEQ ID NO: 296 P297: ELSAT AQEII KSQAI ATKKI aa 4441-4460 SEQ ID NO: 297 P298: TKKII SDYHQ QFRYK LQDFS aa 4457-4476 SEQ ID NO: 298 P299: LQDFS DQLSD YYEKF IAESK aa 4472-4491 SEQ ID NO: 299 P300: IAESK RLIDL SIQNY HTFLI aa 4487-4506 SEQ ID NO: 300 P301: HTFLI YITEL LKKLQ STTVM aa 4502-4521 SEQ ID NO: 301 P302: STTVM NPYMK LAPGE LTIIL aa 4517-4536 SEQ ID NO: 302

The full length sequence of apoB-100 can be found in various publications (such as reference 43, see in particular FIG. 1) herein incorporated by reference in its entirety.

Example 2 ApoB-100 Peptides Selection

Plasma samples were obtained from 10 patients with clinically evident atherosclerotic heart disease. In addition, 50 plasma samples were obtained from 25 men and 25 women with no evidence of atherosclerotic heart disease. Samples of the 20 amino acid long peptides were adsorbed to microtiter plates to perform enzyme-linked immunosorbent assay (ELISA) analyses of the plasma samples. Peptides were used in their native state or after oxidation by exposure to copper or after modification by malondialdehyde (MDA).

Plasma samples from patients and normal subjects contained antibodies to a large number of different peptides. Antibodies against both native and modified peptides were identified. A total of 38 peptide sequences were identified as potential targets for immune reactions that may be of importance for the development of atherosclerosis.

The peptide sequences against which the highest antibody levels were detected could be divided in six groups with certain common characteristics as indicated in Table 2 below.

TABLE 2 ApoB-100 SEQ ID Peptide Sequence Amino Acid NO. A. High levels of IgG antibodies to MDA-modified peptides (n = 3) P11 FLDTV YGNCS THFTV KTRKG 151-170 11 P25 PQCST HILQW LKRVH ANPLL 361-380 25 P74 VISIP RLQAE ARSEI LAHWS 1096-1115 74 B. High levels of IgM antibodies, no difference between native and MDA-modified peptides (n = 9) P40 KLVKE ALKES QLPTV MDFRK 586-605 40 P94 DGSLR HKFLD SNIKF SHVEK 1396-1415 94 P99 KGTYG LSCQR DPNTG RLNGE 1471-1490 99 P100 RLNGE SNLRF NSSYL QGTNQ 1486-1505 100 P102 SLTST SDLQS GIIKN TASLK 1516-1535 102 P103 TASLK YENYE LTLKS DTNGK 1531-1550 103 P105 DMTFS KQNAL LRSEY QADYE 1561-1580 105 P177 MKVKI IRTID QMQNS ELQWP 2641-2660 177 C. High levels of IgG antibodies, no difference between native and MDA-modified peptides (n = 2) P143 IALDD AKINF NEKLS QLQTY 2131-2150 143 P210 KTTKQ SFDLS VKAQY KKNKH 3136-3155 210 D. High levels of IgG antibodies to MDA-modified peptides and at least twice as much antibodies in the plasma of normal subjects as compared to the plasma of individuals with atherosclerosis (n = 5) P1 EEEML ENVSL VCPKD ATRFK  1-20 1 P129 GSTSH HLVSR KSISA ALEHK 1921-1940 129 P148 IENID FNKSG SSTAS WIQNV 2206-2225 148 P162 IREVT QRLNG EIQAL ELPQK 2416-2435 162 P252 EVDVL TKYSQ PEDSL IPFFE 3766-3785 252 E. High levels of IgM antibodies to MDA-modified peptides and at least twice as much antibodies in the plasma of normal subjects as compared to the plasma of individuals with atherosclerosis (n = 11) P30 LLDIA NYLME QIQDD CTGDE 436-455 30 P31 CTGDE DYTYK IKRVI GNMGQ 451-470 31 P32 GNMGQ TMEQL TPELK SSILK 466-485 32 P33 SSILK CVQST KPSLM IQKAA 481-500 33 P34 IQKAA IQALR KMEPK DKDQE 496-515 34 P100 RLNGE SNLRF NSSYL QGTNQ 1486-1505 100 P107 SLNSH GLELN ADILG TDKIN 1591-1610 107 P149 WIQNV DTKYQ IRIQI QEKLQ 2221-2240 149 P169 TYISD WWTLA AKNLT DFAEQ 2521-2540 169 P236 EATLQ RIYSL WEHST KNHLQ 3526-3545 236 P301 HTFLI YITEL LKKLQ STTVM 4501-4520 301 F. High levels of IgG antibodies, but no difference between intact and MDA-modified peptides but at least twice as much antibodies in the plasma of individuals with atherosclerosis as compared to the plasma of normal subjects (n = 7) P10 ALLVP PETEE AKQVL FLDTV 131-150 10 P45 IEIGL EGKGF EPTLE ALFGK 661-680 45 P111 SGASM KLTTN GRFRE HNAKF 1651-1670 111 P154 NLIGD FEVAE KINAF RAKVH 2296-2315 154 P199 GHSVL TAKGM ALFGE GKAEF 2971-2990 199 P222 FKSSV ITLNT NAELF NQSDI 3316-3335 222 P240 FPDLG QEVAL NANTK NQKIR 3586-3605 240 G. No level of IgG or IgM antibodies (n = 1) P2 ATRFK HLRKY TYNYE AESSS 16-35 2

Example 3 ApoB-100-Peptides Selection

Inhibition of atherosclerosis in apo E−/− mice by immunization with fifteen different test articles based on fifteen different peptide fragments of apoB-100 was investigated.

TABLE 3A Peptide Sequence Apolipoprotein B aa SEQ ID NO P2: ATRFK HLRKY TYNYE AESSS aa 16-35 SEQ ID NO: 2 P11: FLDTV YGNCS THFTV KTRKG aa 151-170 SEQ ID NO: 11 P25: PQCST HILQW LKRVH ANPLL aa 361-380 SEQ ID NO: 25 P32: GNMGQ TMEQL TPELK SSILK aa 466-485 SEQ ID NO: 32 P45: IEIGL EGKGF EPTLE ALFGK aa 661-680 SEQ ID NO: 45 P74: VISIP RLQAE ARSEI LAHWS aa 1096-1115 SEQ ID NO: 74 P102: SLTST SDLQS GIIKN TASLK aa 1516-1535 SEQ ID NO: 102 P129: GSTSH HLVSR KSISA ALEHK aa 1921-1940 SEQ ID NO: 129 P143: IALDD AKINF NEKLS QLQTY aa 2131-2150 SEQ ID NO: 143 P148: IENID FNKSG SSTAS WIQNV aa 2206-2225 SEQ ID NO: 148 P154: NLIGD FEVAE KINAF RAKVH aa 2296-2315 SEQ ID NO: 154 P162: IREVT QRLNG EIQAL ELPQK aa 2416-2435 SEQ ID NO: 162 P199: GHSVL TAKGM ALFGE GKAEF aa 2971-2990 SEQ ID NO: 199 P210: KTTKQ SFDLS VKAQY KKNKH aa 3136-3155 SEQ ID NO: 210 P219: ILPSL ELPVL HVPRN LKLSL aa 3271-3290 SEQ ID NO: 219 P240: FPDLG QEVAL NANTK NQKIR aa 3586-3605 SEQ ID NO: 240

In these experiments, apo E−/− mice received primary subcutaneous immunization at 6-7 weeks of age, followed by two boosters administered 3 and 5 weeks later. The mice were fed a high cholesterol diet from 1 week after the second injection (10 weeks of age) and continued until sacrifice at 25 weeks of age. The effect of immunization on atherosclerosis formation was assessed by measuring plaque size (percent area stained with Oil Red O) in an en face preparation of the aorta.

Based on the results from these and other experiments, four peptides were identified as particularly effective in reducing the progression of atherosclerosis.

TABLE 3B Peptide Reduction in atherosclerosis progression P2 (−42%, p < 0.05) P45 (−53%, p < 0.05) P102 (−52%, p < 0.05) P210 (−49%, p = 0.06)

Example 4 Cholera Toxin B Subunit Gene Fusions for Immunization

The gene fusions used in the present disclosure were constructed using a CTB expression vector essentially as described previously (Sadeghi H, Bregenholt S, Wegmann D, Petersen J S, Holmgren J, and Lebens M. Genetic fusion of human insulin B-chain to the B-subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo. Immunology. 2002; 106:237-245). Synthetic oligonucleotides from Innovagen (Lund, Sweden) were used to make double stranded DNA fragments encoding the peptide sequence of interest that could be inserted into the vector such that the added peptide formed a carboxyl extension of mature CTB. The unmodified peptide p210 corresponding to amino acids 3136-3155 of human apoB-100 (KTTKQSFDLSVKAQYKKKNKH—SEQ ID NO:210) was encoded by the DNA sequence:

(SEQ ID NO: 303) 5′ CAAAACGACCAAGCAAAGCTTTGATCTGAGCGTGAAAGCGCAGTATA AGAAAAACAAACACTA 3′ (SEQ ID NO: 304) 5′ CATGGTTTTGCTGGTTCGTTTCGAAACTAGACTCGCACTTTCGCGTC ATATTCTTTTTGTTTGTGATTCGA 3′ (SEQ ID NO: 210) K T T K Q S F D L S V K A Q Y K K N K H

This sequence is 90.9% identical to amino acids 3157-3185 of the murine apoB-100 sequence, the exception being the insertion of a Serine (S) and an Asp (D) residue between N and KH in the C-terminal portion of the peptide:

(SEQ ID NO: 210) Human KTTKQSFDLSVKAQYKKN-KH (SEQ ID NO: 305) Murine KTTKQSFDLSVKAQYKKNSDKH

Oligonucleotides were synthesized that encoded the p210 peptide corresponding to amino acids 3136-3155 of human apoB-100. The coding regions are flanked by sticky ends compatible with restriction enzymes KpnI and HindIII. Insertion into the expression vector leads to an in-frame extension to the carboxyl terminus of mature CTB.

The synthetic sequence was optimized for expression in E. coli. The single strands were annealed and ligated into the pML-CTB vector digested with KpnI and HindIII. Ligated DNA was used to transform E. coli B strain BL21. Transformants were selected initially on the basis of ampicillin resistance and plasmids were then screened using restriction analysis. Finally the sequence of the insert in selected clones was confirmed by DNA sequencing. Protein expression was induced by addition of IPTG to the growth medium. This resulted in the production of insoluble inclusion bodies containing the recombinant protein. The cells were disrupted by sonication following treatment with lysozyme and DNase. The inclusion bodies could be separated from the soluble cell protein and other insoluble cell debris by low speed centrifugation. LPS was removed by washing the inclusion bodies in three times in 0.1% triton X114 in PBS at 4° C. and subsequent extensive washing in PBS in order to remove the detergent. The inclusion bodies were dissolved in 6.5 M urea and reassembled into the biologically active pentameric form by removal of the urea by dialysis against 50 mM sodium carbonate buffer pH 9.0. The assembly and purity of the protein was assessed by SDS-PAGE. Receptor binding activity was confirmed by GM1 ELISA (Svennerholm A M, Holmgren J. Identification of E. coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM1-ELISA) procedure. Curr Microbiol. 1978; 1:19-23). The protein was further partially purified by FPLC gel filtration using a sephadex 200 16/60 column. The OVA-CTB protein used as a control; also a fusion to the carboxyl terminus of mature CTB, was constructed and purified as previously described (George-Chandy A, Eriksson K, Lebens M, Nordstrom I, Schon E, Holmgren J. Cholera toxin B subunit as a carrier molecule promotes antigen presentation and increases CD40 and CD86 expression on antigen presenting cells. Infect Immun. 2001; 69:5716-5725).

Example 5 Mice and Immunization Protocols

Female apoe^(−/−) mice were obtained from Taconic Europe (Ry, Denmark) and Apoe^(−/−) CD4dnTGFβRIItg mice were previously generated (19). Starting at 8 weeks of age, mice were immunized intranasally twice per week for 12 weeks with either p210-CTB or OVA-CTB, both at 15 μg/dose (15 μL volume), or left untreated (PBS). Mice were fed a normal laboratory diet and sacrificed by CO₂ asphyxiation. All experiments were approved by the regional board for animal ethics.

Example 6 Tissue Processing and Immunohistochemistry

Lesion area per cross-section and fractional area of the lesion in the aortic root were quantified and the results expressed as the mean of 5 sections per mouse (22). In brief, fractional lesion area is calculated for each section as F %=(100×L/A) where L is lesion area (μm²) and A is area inside external elastic lamina (μm²). F % is averaged over all levels analyzed (200-600 μm² above aortic cusps) and the mean calculated for each treatment group. This method eliminates artifacts caused by oblique sections.

Primary antibodies (CD4, CD68, VCAM-1, I-A^(b); all rat anti-mouse from BD Biosciences, Franklin Lakes, N.J., U.S.A.) and FoxP3 by eBioscience (San Diego, Calif., U.S.A.) titrated to optimum performance on spleen sections were applied to acetone-fixed cryosections from the aortic root, followed by detection with the ABC alkaline phosphatase kit from Vector Laboratories (Burlingame, Calif., USA). A thresholding technique was implemented using computerized ImagePro analysis (Media Cybernetics, Bethesda, Md., U.S.A.) of immunostained sections. For RNA isolation the thoracic aortic arch distal of the aortic root was dissected and snap-frozen.

Example 7 Flow Cytometry and Intracellular Cytokine Staining

Flow cytometry was performed on a CyAn™ (Dako, Glostrup, Denmark) after staining with the appropriate antibodies; data were analyzed using Summit v4.3 software (Dako). Primary labeled antibodies used were from BD Biosciences (anti-CD4) or from eBioscience (anti-FoxP3). To characterize the cytokine expression profiles of CD4⁺ T cells from lung and spleen of nasally vaccinated mice, cell suspensions were prepared as described before and evaluated by intracellular cytokine staining and FACS analysis. Briefly, lung mononuclear cells were isolated by collagenase Type I digestion (324 U/ml; Sigma) for 1 h on a shaker and splenocytes were prepared by mechanical disruption followed by incubation in erythrocyte lysis buffer (Qiagen, USA) and extensively washed. CD4⁺ T cells were purified using MACS magnetic cell separation as described above. 2×10⁵ spleen or lung cells previously stimulated with 10 μg/ml of human apoB-100 for 24 hours, were restimulated for 4 h at 37° C. in 7,5% CO₂ with PMA (phorbol 12-myristate 13-acetate; 50 ng/ml), ionomycin (1 μg/ml; Sigma) and GolgiPlug (1 μl per 1 ml; BD Bioscience).

Alternatively, 2×10⁵ CD4⁺ T cells previously stimulated with plate bound anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) for 3 days in culture together with recombinant mouse IL-2 (10 ng/ml; Peprotech) and IL-4 (1 ng/ml; Peprotech) followed by a 3 day incubation with only IL-2 and IL-4, were restimulated with plate bound anti-CD3 (5 mg/ml) and anti-CD28 (2 mg/ml) for 5 h in the presence of GolgiPlug. All cells were incubated with FcyR block (BD Bioscience) followed by surface (anti-CD4) and intracellular staining of IFNγ, IL-4, IL-17 or IL-10 (BD Bioscience) and FoxP3 (eBioscience) according to the manufacturer's instructions. Cells were analyzed on a CyAn™ flow cytometer (Dako).

Example 8 Functional Immunoassays

A first group of apoe^(−/−) mice were immunized subcutaneously with HPLC-purified human apoB-100 in complete Freund's adjuvant (CFA) from Pierce (Rockford, Ill., U.S.A.) and boosted 4 weeks later with apoB-100 in incomplete Freund's adjuvant (IFA) from Pierce to generate spleen T cells sensitized to human apoB-100, which were harvested one week later. A second group of mice received the nasal vaccine over 2 weeks (4 doses total/mouse) and CD4⁺ T cells were harvested from the spleen 3 days after the last nasal administration of the vaccine. Spleen CD4⁺ T cells (>95% purity) were isolated by negative selection over a magnetic column using MACS microbeads (CD4+ negative selection kit, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Splenocytes from apoB-100-vaccinated apoe^(−/−) mice were cocultured at varying dilution ratios with purified CD4⁺ T cells from spleens of mice that had received nasal p210-CTB, OVA-CTB or PBS. To exclude contaminating apoB-100 in cell culture media, FCS-free IMDM from Gibco (Invitrogen, Carlsbad, Calif., U.S.A.) was supplemented with ITS™ from BD Biosciences. Cells were incubated for 72 hours in the absence or presence of purified human apoB-100 (20 mg/mL) with incorporation of ³H-thymidine during the last 18 hours. Data are presented as stimulation index (ratio of apoB-100-challenged to unchallenged coculture assay). In a second approach splenocytes and purified CD4⁺ T cells were separated in transwell plates from Corning (Corning, N.Y., U.S.A.) to analyze whether cell-cell contact inhibition abrogated the suppressive effect of tolerized CD4+ T cells.

Example 9 Antibody Assays

ELISA methods were used to quantitate serum Ig isotypes specific for the apoB-100 peptide as well as total IgG and IgM as previously described (19). Sera from immunized mice were tested for antibodies to mouse LDL by incubation (1/50, 1/150 and 1/450 dilutions) in plates coated with mouse LDL (10 μg/ml) and using alkaline phosphatase-conjugated anti-mouse-IgG as detector antibody. Sera from C57BL/6 mice immunized with OVA-CTB were assayed for reactivity to mouse or human LDL, or to apoB-100, at dilutions of 1/25, 1/250 and 1/2500.

Example 10 Real-Time Polymerase Chain Reaction

RNA was isolated from the aortic arch using the RNeasy kit from Qiagen (Hilden, Germany). Total RNA was analyzed by BioAnalyzer from Agilent Technologies (Waldbronn, Germany). Reverse transcription was performed with Superscript-II and random hexamers (both from Invitrogen) and cDNA amplified by real-time PCR using primers and probes for FoxP3, IL-10, TGF-3, IFN-γ and hypoxanthine guanidine ribonucleosyltransferase (HPRT) in an ABI 7700 Sequence Detector from Applied Biosystems. All primers and probes were obtained as “assays on demand” from Applied Biosystems (Foster City, Calif., U.S.A.) Data were analyzed on the basis of the relative expression method with the formula 2^(−ΔΔC) _(T), where ΔΔC_(T)=ΔC_(T) (sample)-ΔC_(T) (calibrator=average C_(T) values of all samples within each group), and ΔC_(T) is the C_(T) of the housekeeping gene (HPRT) subtracted from the C_(T) of the target gene.

Example 11 Serum Analyses

Total serum triglycerides were determined with an enzymatic assay from Roche Diagnostics (Mannheim, Germany) using a TECAN InfiniTE M200 plate reader (TECAN Nordic, Taby Sweden). Total serum cholesterol and lipoprotein profiles were determined by FPLC separation of 2 μL serum of all individuals using a micro-FPLC column from GE Healthcare coupled to a system for online separation and subsequent detection of cholesterol as described, using human serum as reference (Parini P et al., Lipoprotein profiles in plasma and interstitial fluid analyzed with an automated gel-filtration system. Eur J Clin Invest 2006; 36:98-104). IL-10 ELISA from Mabtech (Nacka Strand, Sweden) and TGF-β ELISA from R&D Systems (Minneapolis, Minn., U.S.A.) was used to measure cytokine levels in supernatants.

Example 12 Statistical Analysis

Values are expressed as mean±standard error of the mean (SEM) unless otherwise indicated. Non-parametric Kruskal-Wallis test was used for multiple comparisons, Mann-Whitney U test was used for pairwise comparisons. A p-value of <0.05 was considered significant.

Example 13 Nasal Administration of p210-CTB Inhibits Atherosclerosis

Nasal immunization with p210-CTB caused a significant 35% reduction in atherosclerotic lesion size (p=0.015; p=0.039) and fractional lesion area (p=0.012; p=0.007) in the aortic root as compared with OVA-CTB or untreated controls, respectively (FIG. 1A,B and FIG. 6). Atherosclerosis was not attenuated by administration of OVA-CTB compared with untreated controls indicating an apoB-100 peptide-specific effect (FIG. 1). The composition of the lesions was not significantly altered by p210-CTB immunization, as indicated by quantitative immunohistochemical analysis of markers for CD4⁺ T cells, macrophages (CD68), or the inducible surface proteins I-A^(b) (major histocompatibility complex class II protein) and the vascular cell adhesion molecule-1 (Table 4).

TABLE 4 Weight, cholesterol and triglyceride levels in plasma Cholesterol Triglycerides Group Weight (g) (mg/dL) (mg/dL) apoe^(−/−) P210-CTB 21.0 ± 1.3  281 ± 102 46 ± 9.1 OVA- 21.5 ± 0.6 257 ± 51 36 ± 3.9 CTB Control 20.5 ± 1.0 313 ± 59 52 ± 7.9 apoe^(−/−) × CD4dn P210-CTB 19.1 ± 2.9 254 ± 84 40 ± 8.3 TbRIItg OVA- 20.8 ± 1.2 250 ± 94 40 ± 6.2 CTB p value n.s. n.s. n.s. Mean values and standard deviations are shown. Non-parametric group comparisons were performed using the Kruskal-Wallis test

Example 14 Nasal Administration of p210-CTB does not Affect Plasma Lipids

Immunization did not significantly affect body weight, serum cholesterol or triglycerides (Table 5). Plasma lipoprotein profiles were similar in mice immunized with p210-CTB or OVA-CTB, respectively (FIG. 7).

TABLE 5 Cellular composition and inflammatory markers in aortas of 20 week old apoe^(−/−) mice p P210-CTB (A) OVA-CTB (B) Control value CD4 (cells/mm²) 176 ± 31 151 ± 35  124 ± 21 n.s. I-A^(b) (cells/mm²)  87 ± 25 62 ± 17 112 ± 34 n.s. CD68 (% lesion) 15.9 ± 3.6 22.6 ± 7.4  12.6 ± 2.3 n.s. VCAM-1 (% lesion) 12.7 ± 4.6 22.3 ± 11.3 10.9 ± 1.0 n.s. FOXP3 (cells/mm²)  4.1 ± 0.8 4.2 ± 1.0  6.5 ± 2.6 n.s. Positive cells are calculated per mm² lesion area except for FoxP3 cells, which are presented as cells per total vessel surface area as they were also found in the adventina. Statistics were performed using the non-parametric Kruskall Wallis test

Example 15 CTB Fusion Protein Immunization Increases Aortic FoxP3 and IL-10 mRNA Levels

Real-time reverse transcription-PCR analysis of the thoracic aorta of apoe^(−/−) mice showed significant increases in FoxP3 and IL-10 mRNA levels in both CTB vaccine groups (p210-CTB and OVA-CTB) (FIG. 1C). No statistically relevant differences in FoxP3 or IL-10 mRNA were detected when comparing mice that had received p210-CTB or OVA-CTB, respectively. Furthermore, FoxP3⁺ cell numbers did not differ between groups; a representative example of FoxP3⁺ cells in aortic lesions is shown in FIG. 8. IL-10 was elevated to the same extent in p210-CTB and OVA-CTB groups, pointing to a possible adjuvant effect of CTB. Trends towards increased TGF-β and decreased IFN-γ mRNA in vaccinated mice were not significant.

Example 16 Nasal Vaccination Induces Mucosal and Systemic Humoral and Cellular Immune Responses

P210-CTB immunization induced significantly elevated titers of IgG antibodies to the p210 peptide of apoB-100 (FIG. 2A). Modestly increased IgG anti-p210 was observed in OVA-CTB immunized apoe^(−/−) mice. The IgG1/IgG2a ratio of anti-p210 antibodies did not change, implying that there was no Th1/Th2 shift in T helper activity to B cell activation (FIG. 9 and FIG. 10). Total IgG levels were not influenced by either treatment (FIG. 11). p210-specific IgM titers were significantly elevated both in p210-CTB and OVA-CTB treated groups; however, no difference was detected between p210-CTB and OVA-CTB treated animals (FIG. 2B). Total IgM was not influenced by either treatment (FIG. 12). Sera of immunized mice were tested for antibodies to mouse LDL particles, however, ELISA did not show any such titers (data not shown). Therefore, antibodies induced to human p210 did not recognize intact, endogenous LDL particles in the immunized mice. Apoe^(−/−) mice immunized with OVA-CTB showed modestly increased titers to p210 (FIG. 2). However, parenteral immunization with OVA did not lead to induction of significant IgG antibody responses to mouse LDL (data not shown), thus ruling out serological crossreactivity between OVA and LDL protein.

Analysis of the cellular immune response in the lung, the major organ targeted after nasal vaccination, showed a significant decrease in CD4⁺ T cells expressing interferon-γ (characteristic of Th1 cells) and IL-17 (characteristic of Th17 cells), respectively, in mice treated with p210-CTB (FIG. 3 A,B). In contrast, no such change was recorded for IL-4⁺ CD4⁺ T cells or for FoxP3⁺ CD4⁺ T cells (FIG. 3 C,D). This indicates a shift of the T helper cell balance in the respiratory mucosa, away from the proinflammatory Th1 and Th17 subtypes after nasal immunization with p210-CTB.

Systemic cellular immune responses were monitored in spleen cell preparations. Nasal immunization with p210-CTB significantly increased the proportion of spleen CD4⁺ T cells expressing the anti-inflammatory cytokine IL-10 (FIGS. 4 A and B). Unlike the situation in the lung, no significant differences were detected in the distribution of the remaining CD4⁺ T cell subsets in the spleen, as characterized by intracellular staining for interferon-γ, IL-17, IL-4 and FoxP3 (FIG. 13).

Example 17 P210-CTB Treatment Induces ApoB-100-Specific Treg Activity

To assess whether functional Treg were induced by immunization, we exposed spleen CD4⁺ T cells from apoe^(−/−) mice immunized subcutaneously with human apoB-100 (effector T cells), to CD4⁺ T cells from mice immunized nasally with either p210-CTB, OVA-CTB, or no antigen (FIG. 4C). A marked dose-dependent inhibition of effector T cell proliferation was observed in the presence of CD4⁺ T cells from p210-CTB immunized mice. No such inhibition was observed when T cells from OVA-CTB or non-immunized mice were added. The inhibitory effect of T cells from p210-CTB immunized mice was abolished when these cells were separated from effector T cells by a membrane, indicating that suppression required cell-cell contact (FIG. 14). Levels of 1 L-10 and TGF-β in culture supernatants did not differ between groups (FIG. 15).

Example 18 The Atheroprotective Effect of Nasal p210-CTB Vaccination is Independent of TGF-β Signaling in T Cells

To determine whether the atheroprotective effect of nasal vaccination with p210-CTB depended on TGF-β signaling in T cells, we immunized apoe^(−/−) mice lacking functional TGF-β receptors on T cells (CD4dnTGFβRII×apoe^(−/−) mice). Nasal immunization with p210-CTB significantly reduced atherosclerotic lesion size by 30% in CD4dnTGFβRII×apoe^(−/−) mice, as compared with littermates immunized with OVA-CTB (FIG. 5A and FIG. 16). This indicates that TGFβR signaling in T cells is not required for the atheroprotective effect of nasal p210-CTB vaccination. It also argues against a decisive role for FoxP3+ Treg, as these cells are thought to require TGFβ for their function. IgG but not IgM antibodies directed against the apoB-100 peptide were significantly elevated in all groups of mice immunized with p210-CTB, irrespective of whether signaling via TGF-β was blocked during immunization (FIG. 5B,C). Analysis of mRNA expression in aortas showed no differences in mRNA for, IL-10, TGF-β or interferon-γ between p210-CTB- and OVA-CTB-vaccinated mice (FIG. 17). Surprisingly, FoxP3 mRNA was not reduced in CD4dnTGF β RII×apoe^(−/−) mice, possibly reflecting the presence of immature Treg (FIG. 17). Furthermore, quantitative immunohistochemistry showed no differences in cellular composition of lesions between treatment groups (Table 6).

TABLE 6 Cellular composition and inflammatory markers in aortas of 20 week old apoe^(−/−) CD4dnTGFβRIItg mice P210-CTB (C) OVA-CTB (D) p value CD4 (cells/mm²) 120 ± 43  173 ± 55 n.s. I-A^(b) (cells/mm²) 94 ± 16  238 ± 102 n.s. CD68 (% lesion) 9.9 ± 2.8 14.5 ± 2.6 n.s. VCAM-1 (% lesion) 7.6 ± 2.6 19.3 ± 2.2 n.s. FOXP3 (cells/mm²) 19.7 ± 5.2  25.6 ± 3.4 n.s. Positive cells are calculated per mm² lesion area except for FoxP3 cells, which are presented as cells per total vessel surface area as they were also found in the adventitia. Statistics were performed using the non-parametric Kruskall Wallis test

In the above exemplary procedures, a peptide comprising amino acids 3136-3155 of apoB-100 (p210) was fused to the B subunit of cholera toxin (CTB), which binds to a ganglioside on mucosal epithelia. The effect of nasal administration of the p210-CTB fusion protein on atherogenesis was compared with that of an ovalbumin peptide fused to CTB and with untreated controls. Immunization with p210-CTB for 12 weeks caused a 35% reduction in aortic lesion size of apoe^(−/−) mice. This effect was accompanied by induction regulatory T cells that markedly suppressed effector T cells rechallenged with apoB-100 and increased numbers of IL-10⁺ CD4⁺ T cells. Furthermore, a peptide-specific antibody response was observed. Atheroprotection was also documented in apoe^(−/−) mice lacking functional transforming growth factor-beta receptors on T cells.

The above results confirm and extend previous reports on atheroprotective effects of immunization with LDL or its components (5-8, 12, 15, 16). The use of complete LDL particles as immunogens is not attractive for clinical vaccination strategies since these particles may contain multiple pro-inflammatory and even potentially toxic molecules such as modified lipids and endotoxins. Recent studies have identified a set of apoB-100-derived peptides with significant atheroprotective effects (15, 16), enabling development of a structurally defined vaccine candidate. Among them, specific native peptides were immunogenic in humans and mice and correlated with the extent of atherosclerotic disease (33-34). By combining a limited number of peptides in the vaccine, overcoming MHC restriction is expected. Combining peptide sequences with immunomodulatory components (adjuvants) such as CTB is an attractive approach to selectively induce protective immunity while avoiding side effects caused by non-peptide components in LDL particles. Unlike LDL, the vaccine formulation can be manufactured in a reproducible way and under Good Manufacturing Practice (GMP) conditions. The possibility to induce atheroprotective immunity by nasal administration of an LDL component is also attractive for clinical medicine.

In summary, the present disclosure provides fusion products and related compositions methods and systems that in several embodiments allow performing a strategy for atheroprotective immunization. A peptide sequence from apolipoprotein B-100 of low-density lipoprotein fused with a carrier such as the B subunit of cholera toxin is described and used for immunization of mices and in particular for intranasal immunization of apoe^(−/−) mice. Methods and systems herein described led to antigen-specific regulatory T cells and a 35% reduction of atherosclerosis.

The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the fusion proteins, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Modifications of the above-described modes for carrying out the disclosure that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference.

Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.

It is to be understood that the disclosures are not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein can be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the fusion proteins, fusion protein components, compositions, methods steps, and systems set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.

In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

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1. A fusion protein comprising an antigenic fragment of apoB-100 protein or a derivative thereof and a protein carrier, wherein the antigenic fragment and the protein carrier are comprised in the fusion protein in a 1:1 fragment:carrier molar ratio; and wherein the fusion protein is capable of inducing antigen specific regulatory T cells, the antigen specific regulatory T cells being specific for the antigenic fragment of apoB-100.
 2. The fusion protein of claim 1, wherein the antigenic fragment comprises one or more peptides selected from the group consisting of P2, P11, P25, P32, P45, P74, P102, P129, P143, P148, P154, P162, P210, P219, and P240.
 3. The fusion protein of claim 1, wherein the antigenic fragment comprises one or more peptide selected from the group consisting of P2, P45, P102 and P210.
 4. The fusion protein of claim 1, wherein the antigenic fragment comprises P210.
 5. The fusion protein of claim 1, wherein the protein carrier is selected from the group consisting of Avidin, BTG protein, Bovine G globulin, Bovine Immunoglobulin G, Bovine Thyroglobulin, Bovine Serum Albumin (BSA), Conalbumin, Edestein, Exoprotein A from Pseudomonas aeruginosa, HC (Hemocyanin from crab Paralithodes camtschatica), Helix Promatia Haemocyanin (HPH), Human Serum Albumin (HSA), KTI (Kunits trypsin inhibitor from soybeans), Keyhole Limpet Heamocyanin (KLH), LPH (Haemocyanin from Limulus polyphemus) Ovalbumin, Pam3Cys-Th, Polylysine, porcine Thyroglobulin (PTG), Purified Protein Derivative (PPD), Rabbit Serum Albumin (RSA), Soybean Trypsin Inhibitor (STI), and Sunflower Globulin (SFG).
 6. The fusion protein of claim 1, wherein the protein carrier is a cytokine.
 7. The fusion protein of claim 1, wherein the protein carrier is subunit B of cholera toxin.
 8. A method to treat and/or prevent atherosclerosis in an individual, the method comprising: administering to the individual an effective amount of the fusion protein of claim 1, the effective amount eliciting an antigen specific Treg immunomodulatory response in the individual, the antigen specific Treg immunomodulatory response being specific for the antigenic fragments of apoB-100 or a derivative thereof.
 9. The method of claim 8, wherein the administering is performed via an oral or nasal or nasal route of administration.
 10. The method of claim 8, wherein the administering is performed via a subcutaneous route of administration.
 11. The method of claim 8, wherein the administering is performed via an intramuscular route of administration.
 12. An immunogenic composition comprising the fusion protein of claim 1 together with an adjuvant and/or an excipient.
 13. The immunogenic composition of claim 12 wherein the adjuvant and/or excipients are pharmaceutically acceptable and the composition is a pharmaceutical composition. 